The ability to deliver precise amounts of biomolecules and nanofabricated probes into living cells offers tremendous opportunities for biological studies and therapeutic applications. It may also play a key role in the non-viral generation of engineered stem cells and induce pluripotent stem cells with high efficiency and non-carcinogenic properties.
A variety of cell transfection techniques have been developed including: viral vectors, chemical methods (e.g. complexes with lipids, calcium phosphate, polycations or basic proteins) and physical methods (e.g., particle bombardment, micro-injection and electroporation). Except for micro-injection these techniques are based on bulk stochastic processes where cells are transfected randomly by a large number of genes (>108/cell). A disadvantage of such methods is that the injected dose cannot be controlled.
Classical chemical transfection methods including lipoplex and polyplex based nanoparticles are often inefficient, and the level of cytotoxicity of many chemicals used is still unclear. In comparison, physical approaches are capable of delivering genes safely and efficiently because these methods can directly transfer naked genes into cells. Among them, biolistic transfection (i.e. hand-held gene gun) can be applied to a wide variety of cell/tissue types, but it causes significant physical damages to cells, and gold/tungsten particle carriers may have a negative impact on cell functions. Micro-injection is the most accurate and precise existing tool which has been widely used to generate transgenic animal models for biomedical research. Pronuclear micro-injection, in particular, is to inject a piece of manipulated DNA (also called “transgene”) into one of the pronuclei of mouse donor zygote. The injected zygotes are then transferred into recipient surrogate mice that carry the manipulated embryos to terms. The advantage of such technique is that the gene of
interest is directly and precisely delivered into mammalian cells or specific tissues via a least complicated procedure. Nevertheless, it requires specialized equipment, highly skilled practitioner; the quantity of injected cells is limited within a fixed time, and most unfortunately the efficiency is low. For instance, only about 1% of injected embryos develop into transgenic mice (10-20% of pups born), while transgenic livestock is in the range of 5-10% offspring born. On the other hand, electroporation (EP) is the most widely used method to physically transfect cells because of its technical simplicity, fast delivery, and almost no limitation for cell types and the size of delivery materials. It has been used as a research tool for investigating the biological functions of various potential therapeutic materials in stem cells and in cancer cells. In addition to in vitro study, electroporation is also used as a clinical tool for delivering anticancer drugs (e.g., bleomycin and cisplatin) for cancer therapy and DNA, RNA or DNA vaccines for gene therapy and DNA vaccination.
However, the transport of material is relatively nonspecific resulting in low cell viability and non-uniform transfection.
Bulk electroporation (BEP) is the most widely used physical method to transfect cells because of its technical simplicity, fast delivery, and almost no limitation on cell types and size. Recently, microfluidics-based electroporation (MEP) has emerged as a new technology developed by a number of researchers to transfect individual cells. In MEP, a cell is located next to a small aperture (dimensions of a few micrometers) that focuses the porating electric field to a corresponding area on the cell membrane. MEP offers several important advantages over BEP including lower poration voltages, better transfection efficiency, and a sharp reduction in cell mortality. However, the MEP delivery mechanism is similar to that in BEP: it is diffusion dominated, electric field strengths for the two processes are similar, and for large transfection agents such as nucleic acids or quantum dots, entry into the cytosol is (likely) effected through an attachment onto the outside of the cell membrane followed by an endocytosis-like process. Precise dose control has not been demonstrated using MEP.
None of the aforementioned methods has the ability to deliver a precise amount of therapeutic/detection agents to multiple cells with a wide range of cell size.
One-dimensional nanostructures such as nanochannels (and nanotubes) are characterized by extremely small transverse size and resultant high degree of spatial confinement that endow them a unique set of properties. When patterned laterally, these nanostructures are widely used as critical transport devices for a variety of applications such as sensing, nanomanipulation, and information processing. While numerous fabrication techniques have been developed, few can generate large and highly ordered arrays of both nanochannels and nanowires with no defects and low-cost. The most notable high-resolution lithographic techniques include electron beam lithography (EBL) and focused ion beam milling (FIB), but they are associated with either low throughput or high-cost. Another lithographic technique, nanoimprint lithography (NIL), is of high throughput and relatively low-cost, but it requires use of highly specialized equipment and molds prepared typically by EBL. Many inexpensive techniques have been developed, but they are inadequate in terms of high precision, low defect rate, or large area fabrication of both nanochannels/tubes and nanowires/strands. Moreover, these nanostructures need to be connected to the micro/macro scale structures, such as reservoirs and channels, to form functional devices. This is not a trivial task and the lack of a low-cost solution to this problem significantly limits the applicability of many available nanoconstructs.